The goal of this Phase I SBIR is to investigate the antifungal properties of G4Derm against the complex problem of interkingdom polymicrobial infections in non-healing wounds, utilizing a step-wise approach. Problem to be solved and its magnitude. Non-healing wounds pose a great threat to the public health and burden to the economy. In the United States alone, chronic wounds affect 6-8 million patients and result in an excess of >$25 billion annual health care expenses 1,2. The majority of these costs are related to the treatment of infected DFUs 1. The complexity of the polymicrobial wound bioburden, often harboring multi-drug resistant organisms (MDROs), including pathogenic fungi, further contributes to the chronicity of the wounds and recalcitrance to treatment 3?5. Therefore, there is an urgent need for new antimicrobial approaches that present broad-spectrum activity against both bacteria and fungi to effectively treat chronic wounds. Gap in knowledge the technology will fill. In patients with infected chronic wounds, such as DFUs, effective management of wound bioburden is crucial to prevent severe complications including amputation. Fungal infection is a neglected aspect of chronic wound management, and emerging studies have begun to highlight the need to eliminate both bacterial and fungal pathogens 4. Standard of care does not include antifungal treatment, and pathogens are increasingly developing resistance to conventional antifungals, especially in diabetic patients 6?9. Moreover, solely targeting bacteria in mixed communities results in increased fungal diversity and expansion 10. Additionally, while many antimicrobials are available as wound dressing materials targeting bacteria, they can be toxic to tissue regeneration . For example, antimicrobial hydrogels can clear several strains of bacteria but lack the ability to facilitate host cell proliferation . On the contrary, 20?22 23,24,25 bioengineered skin substitutes promote better tissue regeneration but have numerous challenges such as high costs, limited shelf life, difficult administration, uncontrolled degradation in protease-rich wound environments, and most importantly, device failure due to pathogenic colonization 26,27 because several bacterial and fungal pathogens have an affinity for, and bind to collagen 28?30,31 , thus reserving these expensive products until wound sterility is achieved. Therefore, there is an unmet clinical need for an early, safe, and effective treatment of infected chronic wounds (namely DFUs) that eliminates interkingdom polymicrobial infection and, at the same time, induces wound closure and tissue regeneration. Solution: We propose here a novel self-assembling tissue scaffolding matrix, G4Derm, to (i) prevent/eliminate colonization of infectious pathogens through a unique mechanism of action that is broad spectrum antibacterial and antifungal, and (ii) promote wound closure and tissue regeneration by providing cell attachment sites within the scaffolding matrix. Additionally, G4Derm can gel in situ and conforms to unique wound shapes and depths, thereby enabling easy administration. To establish the feasibility, we propose the following specific aims: Specific aim 1) Demonstrate broad-spectrum antifungal capacity in vitro. Milestones: minimum of 4-log CFU reduction by G4Derm (lead candidate) vs. untreated controls against all fungi tested. Specific aim 2) Demonstrate therapeutic in vivo efficacy of G4Derm to eliminate Candida albicans while promoting wound closure in a diabetic mouse full thickness wound model. Milestones: (a) clearing of C. albicans in infected wounds by < 103 CFU/g of tissue quantified by fungal titers at day 1 comparing average CFU/g of wound tissue treated by G4Derm to untreated control group; (b) wound closure in G4Derm-treated groups compared to untreated control by quantitative optical microscopic, and histopathology assessments at day 21. The results from these studies will enable a Phase II proposal to further evaluate G4Derm?s efficacy against mixed bacterial-fungal pathogens, prevention and treatment of biofilms, GLP/ GMP studies for regulatory approvals, and validation in clinically relevant larger animal models of wound healing while eliminating microbial infections.